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SMAD4 loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells.

Chen YW, Hsiao PJ, Weng CC, Kuo KK, Kuo TL, Wu DC, Hung WC, Cheng KH - BMC Cancer (2014)

Bottom Line: Various studies have proposed potential SMAD4-mediated anti-tumor effects in human malignancy; however, the relevance of SMAD4 in the PDAC molecular phenotype has not yet been fully characterized.Finally, PDAC cells with intact SMAD4 are more sensitive to TGF-β1 inhibitor treatment to reduced cell migration; PDAC cells lacking SMAD4 showed decreased cell motility in response to EGFR inhibitor treatment.This study revealed the molecular basis for SMAD4-dependent differences in PDAC with the aim of identifying the subset of patients likely to respond to therapies targeting the TGF-β or EGFR signaling pathways and of identifying potential therapeutic interventions for PDAC patients with SMAD4 defects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 80424, Taiwan. khcheng@faculty.nsysu.edu.tw.

ABSTRACT

Background: SMAD4 is a gastrointestinal malignancy-specific tumor suppressor gene found mutated in one third of colorectal cancer specimens and half of pancreatic tumors. SMAD4 inactivation by allelic deletion or intragenic mutation mainly occurs in the late stage of human pancreatic ductal adenocarcinoma (PDAC). Various studies have proposed potential SMAD4-mediated anti-tumor effects in human malignancy; however, the relevance of SMAD4 in the PDAC molecular phenotype has not yet been fully characterized.

Methods: The AsPC-1, CFPAC-1 and PANC-1 human PDAC cell lines were used. The restoration or knockdown of SMAD4 expression in PDAC cells were confirmed by western blotting, luciferase reporter and immunofluorescence assays. In vitro cell proliferation, xenograft, wound healing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry analysis were conducted using PDAC cells in which SMAD4 was either overexpressed or knocked down.

Results: Here, we report that re-expression of SMAD4 in SMAD4- PDAC cells does not affect tumor cell growth in vitro or in vivo, but significantly enhances cells migration in vitro. SMAD4 restoration transcriptionally activates the TGF-β1/Nestin pathway and induces expression of several transcriptional factors. In contrast, SMAD4 loss in PDAC leads to increased expression of E-cadherin, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and CD133. Furthermore, SMAD4 loss causes alterations to multiple kinase pathways (particularly the phosphorylated ERK/p38/Akt pathways), and increases chemoresistance in vitro. Finally, PDAC cells with intact SMAD4 are more sensitive to TGF-β1 inhibitor treatment to reduced cell migration; PDAC cells lacking SMAD4 showed decreased cell motility in response to EGFR inhibitor treatment.

Conclusions: This study revealed the molecular basis for SMAD4-dependent differences in PDAC with the aim of identifying the subset of patients likely to respond to therapies targeting the TGF-β or EGFR signaling pathways and of identifying potential therapeutic interventions for PDAC patients with SMAD4 defects.

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SMAD4 reduces E-cadherin, VEGF, EGFR and CD133 expression, but increases TGFβ1/Nestin and CD44 protein levels in PDAC. (A) SMAD4 modulates mRNA levels of EMT and CSC markers in PDAC cells. RT qPCR analyses were performed in AsPC-1 and PANC-1 SMAD4 deficient or proficient cells. Compared to the control, restoration of SMAD4 reduces mRNA level of EGFR, VEGF, CD133 and E-cadherin, but increased Vimentin, SMA, Nestin and CD44 mRNA expression in PDAC. Data were means ± SD of triplicates. *P < 0.05 (B) Western blot analysis of three PDAC cell lines, which had overexpression or knockdown for SMAD4 revealed that the expression levels of CD133, Nestin, EGFR, VEGF and EMT markers in indicated cell lines. β-actin was included as a loading control. (C) Luiferase activity assays for the analysis of CD133 and Nestin transcriptional activities in PDAC cells. Reproter assays were performed using CD133-luc (a) and Nestin-luc(b) reporter constructs in AsPC-1, CFPAC-1 and PANC-1 Smad4 proficient and deficient cells. Bars represent means derived from at least 3 independent experiments. *P < 0.01.
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Figure 3: SMAD4 reduces E-cadherin, VEGF, EGFR and CD133 expression, but increases TGFβ1/Nestin and CD44 protein levels in PDAC. (A) SMAD4 modulates mRNA levels of EMT and CSC markers in PDAC cells. RT qPCR analyses were performed in AsPC-1 and PANC-1 SMAD4 deficient or proficient cells. Compared to the control, restoration of SMAD4 reduces mRNA level of EGFR, VEGF, CD133 and E-cadherin, but increased Vimentin, SMA, Nestin and CD44 mRNA expression in PDAC. Data were means ± SD of triplicates. *P < 0.05 (B) Western blot analysis of three PDAC cell lines, which had overexpression or knockdown for SMAD4 revealed that the expression levels of CD133, Nestin, EGFR, VEGF and EMT markers in indicated cell lines. β-actin was included as a loading control. (C) Luiferase activity assays for the analysis of CD133 and Nestin transcriptional activities in PDAC cells. Reproter assays were performed using CD133-luc (a) and Nestin-luc(b) reporter constructs in AsPC-1, CFPAC-1 and PANC-1 Smad4 proficient and deficient cells. Bars represent means derived from at least 3 independent experiments. *P < 0.01.

Mentions: We and others have shown that SMAD4 is involved in regulating E-cadherin expression in PDAC [8]. One recent study also suggested that SMAD4 is required for TGF-β-induced EMT to mediate bone metastasis of breast cancer cells [23]. Thus, to further confirm that SMAD4 re-expression was involved in alterations of the EMT phenotype marker in PDAC, we performed RT-qPCR and Western blot analysis to evaluate the mRNA and protein levels of EMT-related markers in SMAD4-proficient and -deficient PDAC cells. As shown in Figure 3A, we observed up-regulation of smooth muscle actin and vimentin in the mRNA as well as protein levels and significantly lower levels of E-cadherin in SMAD4-proficient PDAC cells. Meanwhile, pancreatic CSC markers such as CD44, Nestin and CD133 have been shown to play important roles in maintaining PDAC progression. To assess whether SMAD4 re-expression induces alterations in the expression of these CSC markers in PDAC, we further determined the mRNA and protein expression levels of CD44, CD133 and Nestin on SMAD4-deficient and -proficient PDAC cells by RT-qPCR and Western blot analysis. Our Western blot analysis showed that SMAD4-proficient cells express more Nestin and CD44 proteins than SMAD4-deficient cells (Figure 3B). In contrast, the level of CD133 protein expression was reduced in the SMAD4-proficient cells compared to SMAD4- deficient cells (Figure 3B). Additional IHC analysis confirmed a significant increase of E-cadherin, EGFR and CD133 signals and reduced expression of Nestin protein in xenograft tumor samples belonging to PANC-1 shSMAD4 tumors as compared with the control group (Additional file 2: Figure S2). Meanwhile, luciferase reporter assays also confirmed transcriptional regulation of the CD133 and Nestin genes by SMAD4 in PDAC cells (Figure 3C(a and b)).


SMAD4 loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells.

Chen YW, Hsiao PJ, Weng CC, Kuo KK, Kuo TL, Wu DC, Hung WC, Cheng KH - BMC Cancer (2014)

SMAD4 reduces E-cadherin, VEGF, EGFR and CD133 expression, but increases TGFβ1/Nestin and CD44 protein levels in PDAC. (A) SMAD4 modulates mRNA levels of EMT and CSC markers in PDAC cells. RT qPCR analyses were performed in AsPC-1 and PANC-1 SMAD4 deficient or proficient cells. Compared to the control, restoration of SMAD4 reduces mRNA level of EGFR, VEGF, CD133 and E-cadherin, but increased Vimentin, SMA, Nestin and CD44 mRNA expression in PDAC. Data were means ± SD of triplicates. *P < 0.05 (B) Western blot analysis of three PDAC cell lines, which had overexpression or knockdown for SMAD4 revealed that the expression levels of CD133, Nestin, EGFR, VEGF and EMT markers in indicated cell lines. β-actin was included as a loading control. (C) Luiferase activity assays for the analysis of CD133 and Nestin transcriptional activities in PDAC cells. Reproter assays were performed using CD133-luc (a) and Nestin-luc(b) reporter constructs in AsPC-1, CFPAC-1 and PANC-1 Smad4 proficient and deficient cells. Bars represent means derived from at least 3 independent experiments. *P < 0.01.
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Figure 3: SMAD4 reduces E-cadherin, VEGF, EGFR and CD133 expression, but increases TGFβ1/Nestin and CD44 protein levels in PDAC. (A) SMAD4 modulates mRNA levels of EMT and CSC markers in PDAC cells. RT qPCR analyses were performed in AsPC-1 and PANC-1 SMAD4 deficient or proficient cells. Compared to the control, restoration of SMAD4 reduces mRNA level of EGFR, VEGF, CD133 and E-cadherin, but increased Vimentin, SMA, Nestin and CD44 mRNA expression in PDAC. Data were means ± SD of triplicates. *P < 0.05 (B) Western blot analysis of three PDAC cell lines, which had overexpression or knockdown for SMAD4 revealed that the expression levels of CD133, Nestin, EGFR, VEGF and EMT markers in indicated cell lines. β-actin was included as a loading control. (C) Luiferase activity assays for the analysis of CD133 and Nestin transcriptional activities in PDAC cells. Reproter assays were performed using CD133-luc (a) and Nestin-luc(b) reporter constructs in AsPC-1, CFPAC-1 and PANC-1 Smad4 proficient and deficient cells. Bars represent means derived from at least 3 independent experiments. *P < 0.01.
Mentions: We and others have shown that SMAD4 is involved in regulating E-cadherin expression in PDAC [8]. One recent study also suggested that SMAD4 is required for TGF-β-induced EMT to mediate bone metastasis of breast cancer cells [23]. Thus, to further confirm that SMAD4 re-expression was involved in alterations of the EMT phenotype marker in PDAC, we performed RT-qPCR and Western blot analysis to evaluate the mRNA and protein levels of EMT-related markers in SMAD4-proficient and -deficient PDAC cells. As shown in Figure 3A, we observed up-regulation of smooth muscle actin and vimentin in the mRNA as well as protein levels and significantly lower levels of E-cadherin in SMAD4-proficient PDAC cells. Meanwhile, pancreatic CSC markers such as CD44, Nestin and CD133 have been shown to play important roles in maintaining PDAC progression. To assess whether SMAD4 re-expression induces alterations in the expression of these CSC markers in PDAC, we further determined the mRNA and protein expression levels of CD44, CD133 and Nestin on SMAD4-deficient and -proficient PDAC cells by RT-qPCR and Western blot analysis. Our Western blot analysis showed that SMAD4-proficient cells express more Nestin and CD44 proteins than SMAD4-deficient cells (Figure 3B). In contrast, the level of CD133 protein expression was reduced in the SMAD4-proficient cells compared to SMAD4- deficient cells (Figure 3B). Additional IHC analysis confirmed a significant increase of E-cadherin, EGFR and CD133 signals and reduced expression of Nestin protein in xenograft tumor samples belonging to PANC-1 shSMAD4 tumors as compared with the control group (Additional file 2: Figure S2). Meanwhile, luciferase reporter assays also confirmed transcriptional regulation of the CD133 and Nestin genes by SMAD4 in PDAC cells (Figure 3C(a and b)).

Bottom Line: Various studies have proposed potential SMAD4-mediated anti-tumor effects in human malignancy; however, the relevance of SMAD4 in the PDAC molecular phenotype has not yet been fully characterized.Finally, PDAC cells with intact SMAD4 are more sensitive to TGF-β1 inhibitor treatment to reduced cell migration; PDAC cells lacking SMAD4 showed decreased cell motility in response to EGFR inhibitor treatment.This study revealed the molecular basis for SMAD4-dependent differences in PDAC with the aim of identifying the subset of patients likely to respond to therapies targeting the TGF-β or EGFR signaling pathways and of identifying potential therapeutic interventions for PDAC patients with SMAD4 defects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 80424, Taiwan. khcheng@faculty.nsysu.edu.tw.

ABSTRACT

Background: SMAD4 is a gastrointestinal malignancy-specific tumor suppressor gene found mutated in one third of colorectal cancer specimens and half of pancreatic tumors. SMAD4 inactivation by allelic deletion or intragenic mutation mainly occurs in the late stage of human pancreatic ductal adenocarcinoma (PDAC). Various studies have proposed potential SMAD4-mediated anti-tumor effects in human malignancy; however, the relevance of SMAD4 in the PDAC molecular phenotype has not yet been fully characterized.

Methods: The AsPC-1, CFPAC-1 and PANC-1 human PDAC cell lines were used. The restoration or knockdown of SMAD4 expression in PDAC cells were confirmed by western blotting, luciferase reporter and immunofluorescence assays. In vitro cell proliferation, xenograft, wound healing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry analysis were conducted using PDAC cells in which SMAD4 was either overexpressed or knocked down.

Results: Here, we report that re-expression of SMAD4 in SMAD4- PDAC cells does not affect tumor cell growth in vitro or in vivo, but significantly enhances cells migration in vitro. SMAD4 restoration transcriptionally activates the TGF-β1/Nestin pathway and induces expression of several transcriptional factors. In contrast, SMAD4 loss in PDAC leads to increased expression of E-cadherin, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and CD133. Furthermore, SMAD4 loss causes alterations to multiple kinase pathways (particularly the phosphorylated ERK/p38/Akt pathways), and increases chemoresistance in vitro. Finally, PDAC cells with intact SMAD4 are more sensitive to TGF-β1 inhibitor treatment to reduced cell migration; PDAC cells lacking SMAD4 showed decreased cell motility in response to EGFR inhibitor treatment.

Conclusions: This study revealed the molecular basis for SMAD4-dependent differences in PDAC with the aim of identifying the subset of patients likely to respond to therapies targeting the TGF-β or EGFR signaling pathways and of identifying potential therapeutic interventions for PDAC patients with SMAD4 defects.

Show MeSH
Related in: MedlinePlus